Mahmood-M-Ali/FlowIC50: FlowIC50 v3.0 — Major Update with New Analysis Tools

Release Notes: FlowPath Dynamics v3.0 This major release marks a significant evolution of the FlowPath Dynamics platform, introducing a multi-channel analysis engine, integrated absolute cell quantification, and a more rigorous statistical framework for dose-response profiling. Major New Features Multi-Channel Generic Analysis Engine Combinatorial Gating: Users can now define multiple markers simultaneously. The application automatically calculates frequencies for all 2^N possible phenotype combinations. Marker-Specific Thresholding: Interactive histograms for each channel allow for precise positivity thresholding with real-time density feedback. Absolute Cell Quantification Volumetric Counting: Full integration of bead-based absolute cell counting (compatible with Precision Count Beads™). Normalized Potency: The engine calculates both traditional phenotypic percentages and absolute cell concentrations (cells/µL). Relative Survival: A new metric that captures total cell loss by normalizing absolute survival values to the untreated control. Dynamic Analysis and Interactive QC Live Engine: Results, curve fits, and plots recalculate instantly when settings are adjusted, eliminating the need to re-process raw FCS files. Manual Data Exclusion: Users can now select specific replicates in the Results dashboard to exclude them from the statistical model with immediate visual feedback. Automated Outlier Detection: Implementation of the Grubbs test (alpha = 0.05) to flag statistically significant outliers for investigator review. Advanced Statistical Framework Logarithmic Inference: All ANOVA tests are now performed on Log10-transformed potency values (pIC50/pEC50), satisfying the normality assumptions of pharmacological data. Post-Hoc Testing: Integrated Tukey's Honest Significant Difference (HSD) test for statistically valid pairwise comparisons between cell lines. Scientific Visualization and Reporting Therapeutic Window Analysis: Implementation of Dumbbell plots to visualize the margin between potency (IC50) and toxicity (LD50). Expression Distribution: Automated generation of Violin and Box plots for comparing Mean Fluorescence Intensity (MFI) shifts across samples. Technical Methodology Appendix: The downloadable HTML report now includes a 20+ page transparency section documenting every algorithm, from Ray-Casting polygon gating to 4-parameter log-logistic modeling. Mathematical Notation: Standardized the display of small concentrations using professional mathematical formatting (e.g., 3.50 x 10^-3) across all tables and plots. Improvements and Bug Fixes Report Resolution: Increased the canvas dimensions for quality control plots to 14" x 12" to prevent distortion in large clinical datasets. Gating Audit Trail: Both the UI and reports now display the complete sequential gating strategy, including bead identification, morphological gates, and marker histograms. Compensation Accuracy: Resolved a matrix handling issue to ensure correct application of Spillover versus Inverse Compensation matrices. Export Robustness: Fixed a ZIP generation failure caused by duplicate column names in multi-marker analysis. Infrastructure: Fully updated Docker configuration for high-performance deployment on cloud platforms like Hugging Face Spaces. Development and Citation Developed by Mahmood Mohammed Ali at Université Grenoble Alpes, Institute for Advanced Biosciences (IAB), Epigenetics of Regeneration and Cancer Group. Software DOI: 10.5281/zenodo.17872796